Cheng Chang

Continuous Production of Lipase-Catalyzed Biodiesel in a Packed-Bed Reactor: Optimization and Enzyme Reuse Study

An optimal continuous production of biodiesel by methanolysis of soybean oil in a packed-bed reactor was developed using immobilized lipase (Novozym 435) as a catalyst in a tert-butanol solvent system. Response surface methodology (RSM) and Box-Behnken design were employed to evaluate the effects of reaction temperature, flow rate, and substrate molar ratio on the molar conversion of biodiesel. The results showed that flow rate and temperature have significant effects on the percentage of molar conversion. On the basis of ridge max analysis, the optimum conditions were as follows: flow rate 0.1 mL/min, temperature 52.1°C, and substrate molar ratio 1 : 4. The predicted and experimental values of molar conversion were 83.31 ± 2.07% and 82.81 ± .98%, respectively. Furthermore, the continuous process over 30 days showed no appreciable decrease in the molar conversion. The paper demonstrates the applicability of using immobilized lipase and a packed-bed reactor for continuous biodiesel synthesis.

Female-specific doublesex dsRNA interrupts yolk protein gene expression and reproductive ability in oriental fruit fly, Bactrocera dorsalis (Hendel).

A homologue of the doublesex gene (Bddsx) has been cloned from the oriental fruit fly, Bactrocera dorsalis (Hendel). Northern analysis indicates a differential expression of Bddsx in male and female flies, as reported for other dsx genes. A structural conservation of DNA binding domain/oligomerization domain 1 and oligomerization domain 2 suggests that the doublesex protein (BdDSX) of this fruit fly serves as a transcriptional factor for downstream sex-specific gene expression. The putative transformer/transformer-2 protein binding sequence in female-specific transcript suggests that a preserved alternative splicing process found in other flies mediates the synthesis of Bddsx transcript. RNA interference (RNAi) data from adult abdominal dsRNA injection assays indicate that female-specific dsx dsRNA reduces specifically its own transcript, inhibits selectively expression of the yolk protein gene (Bdyp1), and delays ovary development. The number of matured eggs is significant reduced after RNAi treatment, but the sex ratio of offspring is not biased. Moreover, 27% of female progeny with RNAi show deformed ovipositor, but male flies are not affected. Although this is a transient treatment, the specific Bddsx(f) interference offers a promising and novel approach to oriental fruit fly control in the future.

Optimization of ultrasound-accelerated synthesis of enzymatic caffeic acid phenethyl ester by response surface methodology

The ultrasound-accelerated enzymatic synthesis of caffeic acid phenethyl ester (CAPE) from caffeic acid and phenethyl alcohol was investigated in this study. A commercial immobilized lipase from Candida antarctica, called Novozym® 435, was used as the catalyst. A 5-level-4-factor central-composite rotatable design (CCRD) and response surface methodology (RSM) were employed to evaluate the effects of reaction time, substrate molar ratio, enzyme amount, and ultrasonic power on percent molar conversion of CAPE. The results indicated that reaction time, substrate molar ratio, and ultrasonic power significantly affected percent molar conversion, whereas enzyme amount did not. A model for synthesis of CAPE was established. Based on ridge max analysis, the optimum condition for CAPE synthesis was predicted to be reaction time 9.6 h, substrate molar ratio 1:71, enzyme amount 2938 PLU, and ultrasonic power 2 W/cm(2) with the molar conversion value of 96.03 ± 5.18%. An experiment was performed under this optimal condition and molar conversion of 93.08 ± 0.42% was obtained.

Responses of striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae), from Taiwan to a range of insecticides

BACKGROUND Information on the insecticide susceptibility of striped stem borer, Chilo suppressalis (Walker), is essential for an effective pest management programme. An early detection of resistance development can prompt the modification of current control methods and increase the lifespan of insecticides through the rotation of chemicals with different modes of action. In this study, the susceptibility of this pest in Taiwan to four classes of insecticides has been examined. RESULTS Over 1000-fold resistance to carbofuran was detected in C. suppressalis collected from Chiayi and Changhua prefectures, with estimated LC(50) values of > 3 mg cm(-2). In addition, 61-fold resistance to cartap was found in the Chiayi population. On the other hand, all tested populations of rice stem borer were still relatively susceptible to chlorpyrifos, fipronil and permethrin, with LC(50) values ranging from 30 to 553 ng cm(-2). Chilo suppressalis populations collected from the central parts of Taiwan have a higher degree of resistance to the tested insecticides than those from northern areas. CONCLUSION The occurrence of high resistance to carbofuran in the Chiayi and Changhua areas suggests that this compound should be replaced with chemicals having a different mode of action, such as chlorpyrifos, fipronil and permethrin, to which low cross-resistance has been detected.

Optimization of lipase-catalyzed biodiesel by isopropanolysis in a continuous packed-bed reactor using response surface methodology

Isopropanolysis reactions were performed using triglycerides with immobilized lipase in a solvent-free environment. This study modeled the degree of isopropanolysis of soybean oil in a continuous packed-bed reactor when Novozym 435 was used as the biocatalyst. Response surface methodology (RSM) and three-level-three-factor Box-Behnken design were employed to evaluate the effects of synthesis parameters, reaction temperature ( degrees C), flow rate (mL/min) and substrate molar ratio of isopropanol to soybean oil, on the percentage molar conversion of biodiesel by transesterification. The results show that flow rate and temperature have a significant effect on the percentage of molar conversion. On the basis of ridge max analysis, the optimum conditions for synthesis were as follows: flow rate 0.1 mL/min, temperature 51.5 degrees C and substrate molar ratio 1:4.14. The predicted value was 76.62+/-1.52% and actual experimental value was 75.62+/-0.81% molar conversion. Moreover, continuous enzymatic process for seven days did not show any appreciable decrease in the percent of molar conversion (75%). This work demonstrates the applicability of lipase catalysis to prepare isopropyl esters by transesterification in solvent-free system with a continuous packed-bed reactor for industrial production.

Optimized enzymatic synthesis of caffeic acid phenethyl ester by RSM.

In this study, optimization of enzymatic synthesis of caffeic acid phenethyl ester (CAPE), catalyzed by immobilized lipase (Novozym 435) from Candida antarctica was investigated. Novozym 435 was used to catalyze caffeic acid and 2-phenylethanol in an isooctane system. Response surface methodology (RSM) and 5-level-4-factor central-composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction temperature (30-70 degrees C), reaction time (24-72 hours), substrate molar ratio of caffeic acid to 2-phenylethanol (1:10-1:90) and enzyme amounts (100-500 PLU) on percentage conversion of CAPE by direct esterification. Reaction temperature and time had significant effects on percent conversion. On the basis of ridge max analysis, the optimum conditions for synthesis were: reaction time 59 hours, reaction temperature 69 degrees C, substrate molar ratio 1:72 and enzyme amount 351 PLU. The molar conversion of predicted values and actual experimental values were 91.86+/-5.35% and 91.65+/-0.66%, respectively.

Display female-specific doublesex RNA interference in early generations of transformed oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most destructive pests in many Asian countries. An effective strategy to reduce fly density in the field is urgently required. Recently, the doublesex of B. dorsalis (Bddsx(f) ) has been cloned, and RNA interference (RNAi) indicates that it can reduce the offspring in vitro. In this study, a piggyBac-based construct that generates short hairpin RNA (shRNA) against the female-specific region of Bddsx was introduced into the pest to test the RNAi effects on reproductive functions in vivo. RESULTS After embryonic injection and backcross, 21 transgenic lines with germline transformation were identified. Genomic DNA analysis showed that the exogenous transgene including short hairpin Bddsx(f) and a DsRed marker had integrated into the genomes of 11 transformed lines. Northern blot analysis indicated the presence of Bddsx(f) short interfering RNA (siRNA) under the control of a U6 promoter in transformed flies. As expected, the specific effects of RNAi led to the delay of egg maturation, and the offspring was significantly reduced. Reverse transcription real-time PCR further demonstrated that in vivo interference not only specifically inhibited the Bddsx(f) transcript but also repressed expression of the downstream yolk protein gene (Bdyp1). CONCLUSION The results clearly indicate that RNAi is heritable through the expression of specific siRNA in early generations of transformed oriental fruit fly. These results can broaden the understanding of sex-related developmental mechanisms in the fly, and also offer a possible molecular approach for pest control in the future.

Amino acid substitutions of acetylcholinesterase associated with carbofuran resistance in Chilo suppressalis

BACKGROUND Over 1000-fold carbofuran resistance has been observed in Chilo suppressalis (Walker) collected from the Changhua (CH) and Chiayi (CY) prefectures of Taiwan. An understanding of the pertinent mechanisms will benefit effective insecticide resistance management of C. suppressalis. RESULTS Among the five amino acid substitutions of acetylcholinesterase (AChE) identified in C. suppressalis, A314S and H668P had been reported and E101D, F402V and R667Q were novel. Substitution frequencies in AChE of CH and CY populations were much higher than in the susceptible Hsinchu (HC) population. Significantly negative correlations were observed between the frequencies of E101D, A314S and R667Q and the kinetic parameters of AChEs in these populations. AChE from the resistant CH population was less susceptible to the inhibition of carbofuran, with an I50 that was 3.6-fold higher than that of the susceptible HC population. Although Km and Vmax of AChE from the CH and CY populations were reduced to 72-87% of those from the HC population, the overall catalytic efficiency (Vmax /Km ) remained constant for all three populations. CONCLUSION Amino acid substitutions identified in the AChE of C. suppressalis are associated with changes in AChE kinetics and its insensitivity to carbofuran. These observations are helpful for rapid monitoring, prediction and management of OP and CB resistance in the field.

Optimal α-Chymotrypsin-Catalyzed Synthesis of N-Ac-Phe-Gly-NH2

N-Acetyl-phenylalanine-glycinamide (N-Ac-Phe-Gly-NH(2)), a type of dipeptide derivative, was synthesized from N-acetyl phenylalanine ethyl ester and glycinamide and catalyzed by alpha-chymotrypsin, a protease, in a biphasic system. Response surface methodology with a four-factor, five-level central composite rotatable design was employed to evaluate the effects of selected parameters that included incubation time, reaction temperature, enzyme activity, and pH level on the yield of the dipeptide derivative. The results indicated that pH significantly affected the yield of N-Ac-Phe-Gly-NH(2). In a ridge max analysis, the optimum condition for this synthesis included an incubation time of 30.9 min, a reaction temperature of 35.8 degrees C, an enzyme activity of 159.2 U, and a pH of 8.98. The predicted and the actual (experimental) yields were 98.0 and 95.1%, respectively.

Optimization of ultrasound‐accelerated synthesis of enzymatic octyl hydroxyphenylpropionate by response surface methodology

The ultrasound‐accelerated enzymatic synthesis of octyl hydroxyphenylpropionate (OHPP) from p‐hydroxyphenylpropionic acid (HPPA) and octanol was investigated in this study. A commercially available immobilized lipase from Candida antarctica, Novozym® 435, was used as the biocatalyst. A three‐level‐three‐factor Box‐Behnken design experiment and response surface methodology were used to evaluate the effects of temperature, reaction time, and enzyme activity on percent yield of OHPP. The results indicated that temperature and enzyme activity significantly affected percent yield, whereas reaction time did not. A model for the synthesis of OHPP was established. Based on a ridge max analysis, the optimum conditions for OHPP synthesis were predicted to use a reaction temperature of 58.8°C, a reaction time of 14.6 h, and an enzyme activity of 410.5 PLU with a yield of 98.5%. A reaction was performed under these optimal conditions, and a yield of 97.5% ± 0.1% was obtained.